The determination of the three-dimensional structure of ds (double stranded)-DNA, RNA and other non-traditional, intact nondenatured nucleic acids is extremely important for the understanding of their interactions with proteins as well as other biomolecules and nonbiomolecules involved in prokaryotic and eukaryotic regulation, gene expression and repair, among other fields. See Sanger, W. Principles of Nucleic Acid Structure. 1984, Springer-Verlag, NewYork; Sinden, R. R., 1994, DNA Structure and Function. pp. 1-398. Academic Press, NewYork; Sarma, R. H. Nucleic Acid Geometry and Dynamics. 1980, pp. 1-424. Pergamon Press; Zimmerman, S. B. Ann. Rev. Biochem. 1982; 51:395-427; Dickerson, R. E. 1983. Scientific American, 249(6):94-111, Dickerson, R. E. Methods in Enzym. 211:67-111, 1992; Rich, A. Ann. Rev. Biochem. 53:791-846, 1984). It is also important for the understanding of nondenatured nucleic acid reactions with molecules, such as drugs, that may block the correct reading of the nucleic acid molecule. Ds-DNA can undergo conformational fluctuations, such as helical changes resulting in changes from B- to Z-DNA. Curvature and bending of ds-DNA, and other nondenatured nucleic acid molecules can take place alone or when the molecules are complexed with other substances, such as a protein or plurality of proteins. Protein-DNA complexes can cause major conformational changes in the ds-DNA or RNA, such as sharp kinks and bending toward the major groove (J. Kim et al., 1993 Nature 365:512-520; J. L. Kim et al., 1993 Nature 365:520-527). For instance, human 170 KDa topoisomerase II binds preferentially to curved left-handed Z-DNA (J. Biomol. Struc. & Dyn. 12:605-623, 1994). Variant states and structures of DNA have biological and physical properties that differ from those of bulk DNA. Examples of such variant states include sites of base mismatches, DNA bulges, DNA-histone complexes, DNA sequences that induce bending of the helix axis, cruciforms, H-DNA and other branched species (Cooper, J. P. et al, Proc. Natl. Acad., Sci. USA 86:7336-7340, 1989), and so-called left-handed Z-DNA. Some of these states have been implicated in biological functions including mutagenesis, control of transcription and/or gene expression), or genetic recombination.
One of the approaches to assessing the biological role of high-energy forms of ds-DNA, such as Z-DNA, is to reveal such ambient conditions (e.g., pH, ionic strength, temperature, milieu polarity, high pressure, photodynamic damage, as would favor stabilization of alternative DNA (and other nucleic acid structures. Such studies, however, require a source of stable, immobilized molecules which can then be used for further study.
Microarrays, or solid phase structures containing hundreds or thousands of molecules affixed thereto, have become an important feature of molecular biology, especially in areas like genomics, and in screening arrays; however, the methods available to this point in time for immobilizing molecules generally, and nucleic acid molecules in particular, are limited. Especially limited are methods for immobilizing somewhat more uncommon form of nucleic acids including those which are in, e.g., Z conformation, multistranded molecules, such as nucleic acid molecules containing 2-10, preferably 2-6 strands and circular molecules, like plasmids, which may be supercoiled in whole or in part.
It is the aim of this invention to provide methods and apparatus that are useful in immobilizing such molecules, as well as assays which can be used with the resulting molecules.
How these features of the invention are achieved will be seen in the detailed disclosure which follows.